Fluorescence lifetime FRET assay for live-cell high-throughput screening of the cardiac SERCA pump yields multiple classes of small-molecule allosteric modulators

We have used FRET-based biosensors in live cells, in a robust high-throughput screening (HTS) platform, to identify small-molecules that alter the structure and activity of the cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA2a). Our primary aim is to discover drug-like small-molecule activators that improve SERCA’s function for the treatment of heart failure. We have previously demonstrated the use of an intramolecular FRET biosensor, based on human SERCA2a, by screening two different small validation libraries using novel microplate readers that detect the fluorescence lifetime or emission spectrum with high speed, precision, and resolution. Here we report results from FRET-HTS of 50,000 compounds using the same biosensor, with hit compounds functionally evaluated using assays for Ca2+-ATPase activity and Ca2+-transport. We focused on 18 hit compounds, from which we identified eight structurally unique scaffolds and four scaffold classes as SERCA modulators, approximately half of which are activators and half are inhibitors. Five of these compounds were identified as promising SERCA activators, one of which activates Ca2+-transport even more than Ca2+-ATPase activity thus improving SERCA efficiency. While both activators and inhibitors have therapeutic potential, the activators establish the basis for future testing in heart disease models and lead development, toward pharmaceutical therapy for heart failure.

(1) FLT-FRET HTS of 50K DIVERSet Library, to identify initial hit compounds  19 . Here we focus on hits that decrease FRET (increase FLT). (B) Screening funnel describing the 5-step process in this study, involving measurements of FLT-FRET and SERCA function, with SERCA in live mammalian cells (HEK293) and in isolated pig cardiac SR membranes, respectively: (1) FLT changes caused by test compounds were measured using the SERCA-specific FRET biosensor 2CS (two-color SERCA), to identify 2960 initial hit compounds. False hits were ruled out as compounds that (2) are fluorescent or affect the donor directly, decreasing the hit compounds to 295, (3) decrease FLT (increase FRET), reducing the hit compounds to 160, and (4) affect FRET in a null-biosensor, in which donor and acceptor are separated by a nonfunctional flexible peptide, reducing the hit compounds to 91, of which 18 were selected for step 5 (see Results under "Removal of null-biosensor effectors"). (5) Compound concentration-dependence of FRET and SERCA function was measured to further prioritize hit compounds for future lead development. Experimental details are provided in Methods. www.nature.com/scientificreports/ In previous early-stage drug discovery campaigns, we focused on the SERCA regulator, PLB, via intermolecular FRET biosensor designs 23,24 . We have also validated intramolecular FRET biosensor constructs of SERCA 20,22,25 , to detect binding of compounds directly to SERCA. For this, we engineered a "two-color" SERCA (2CS, Fig. 1A) construct with eGFP and tagRFP fluorescent proteins fused to the cytoplasmic N-and A-domains of SERCA, to detect relative motions of these domains during the enzymatic cycle responsible for Ca 2+ -transport 22,25,26 . Measuring FRET within 2CS, stably expressed in a mammalian cell line (HEK293), we previously validated this biosensor using the NCC (727 compounds) 22 and LOPAC (1280 compounds) 25 libraries. The next logical step, in the present study, is to use 2CS in HTS of a 50,000-compound DIVERSet library, a diverse collection of drug-like small molecules that has yielded effective hit compounds in other drug discovery projects [27][28][29] . HTS was enabled by the FLT-PR (fluorescence lifetime plate reader), which scans a 1536-well plate with unprecedented precision and speed, determining FLT with ~ 0.3% CV (30 times better precision than conventional intensity detection) in 2.5 min 23,27,28 , making possible a high-precision 50,000-compound screen in 2 days. To remove false positives, a spectral unmixing plate reader (SUPR) was used to provide complementary spectral measurement of compound-induced FRET changes 26 .
Although other biosensors are under development (e.g., using orange and maroon fluorescent proteins) 22,23,26 , the GFP/RFP 2CS biosensor has been thoroughly validated for screening the DIVERSet compound library (DIVERSet-CL) using HEK293 cells. To validate selected hit compounds and prioritize those with lead potential, we acquired concentration response curves (CRCs) using FRET and functional assays (Ca 2+ -ATPase activity and Ca 2+ -uptake). We hypothesized that the combination of improved fluorescence technology and screening a larger library of compounds would yield a larger and more diverse collection of hit compounds that improve cardiac SERCA function, thus increasing the potential for discovering lead compounds for new heart failure therapeutics.
Compounds that significantly altered the structure of 2CS were determined from their change in lifetime (Δτ) vs DMSO controls (2CS plus DMSO), and Δτ was compared to the normal statistical fluctuation of the biosensor by computing the robust (r)Z-score (see Methods). FLT changes induced by potential hit compounds (Fig. 2B, red) are distinct from the normal distributions of DMSO controls (Fig. 2C, dark blue) and of compounds not affecting SERCA2a (Fig. 2C, light blue). A hit threshold was set at rZ-score = ± 3, resulting in 2960 initial hit compounds (Fig. 2B, step 1), which is ~ 10 × more initial hit compounds than we previously identified with an FLT HTS of the DIVERSet library of 50,000-compounds, using the approach described in Fig. 1A to identify initial hit compounds. (A) Screening precision was determined by computing %CV for each plate using DMSO control wells, with a median value of 0.4% across 40 plates. (B) The change in lifetime (Δτ) was computed to find potential hits (red) with hit threshold set at rZ-score = ± 3, resulting in 2960 initial hits for triage with the SUPR instrument. DMSO controls (dark blue) and compounds not affecting 2CS (light blue) are grouped in the plot to illustrate plate boundaries. (C) The histogram of compounds not affecting 2CS (light blue, 1 ps bin width) shows normal distribution, similar to that of DMSO controls (no compound), as shown by a fit of the populations to Gaussian distributions. The horizontal lines in (B,C) illustrate the approximate cutoffs used, though actual cutoffs were determined on a plate-by-plate basis. www.nature.com/scientificreports/ ATPase-based HTS assay 30 using a similar threshold. Optically interfering compounds were removed (Fig. 1B, step 2) using a spectral unmixing plate reader to obtain spectra for 2CS that were analyzed relative to the donoronly 1CS sample 22 (SUPR, see Methods). 295 compounds remained. We cannot predict the direction of ΔFLT for activator vs. inhibitor. However, FLT increasers were preferred because: (a) more FLT decreasers were found to fail these tests 23,25,26,31 , (b) increasers offer greater reproducibility 23,25,26 , and (c) most previously identified SERCA modulators have been shown to be FLT increasers 20,22,25 . Therefore, we prioritized 160 FLT increasers (termed "hit compounds" (Fig. 1B, step 3) for retesting with a null-biosensor, to remove false positives.
Removal of null-biosensor effectors. 160 hit compounds were retested (Fig. 1B, step 4) using 2CS ( Fig. 3A and C; Supplementary Fig. S1) and a null-biosensor ( Fig. 3B and D), GFP and RFP connected by a 32-residue unstructured flexible linker peptide (G32R) 25 , to rule out compounds that directly bind to the fluorescent proteins and alter FLT. ΔFLT (Δτ) from the FLT-PR and Δ(G/R) (change in the ratio of mole fractions of donor [green, G] and acceptor [red, R] in the emission spectrum as determined from a linear combination of component spectra of donor-only and acceptor-only (1CS) samples 22 ) from the SUPR were determined for each compound. As these are two complementary measures of FRET (FRET decreases both), a strong correlation was observed in 2CS for compounds that induced a structural change much greater than observed in the nullbiosensor ( Fig. 3C and D) (see Methods under "FRET-HTS instrumentation and data analysis").
Hit compounds that produced Δτ ≥ 70 ps (91 compounds, Fig. 1B, step 4; Fig. 3A, Supplementary Fig. S1A), but excluding any that exceeded a 50 ps response in the null-biosensor, were targeted for further functional testing. After determining compound availability for repurchase, we selected 18 compounds with a representative range of ΔFLT ( Supplementary Fig. S1B) for CRC testing. None of these compounds were in the PAINS (Pan-Assay INterference compoundS) category 32 , nor were they redox agents or metal chelators.   Fig. 1B were then selected and dispensed into 1536-well plates containing 10 and 30 μM [compound] (n = 3 wells for each concentration), using the same compound stock solutions as in the HTS phase (step 1 in Fig. 1B). These repeat readings establish the reproducibility needed to select a set of hit compounds for re-purchase as solids, for subsequent studies discussed below. Data are shown from the 30 μM wells for 2CS (A,C) and null-biosensor (B,D). (A) Distribution of significant ΔFLT for the 2CS biosensor. (B) The 160 hit compounds were counter-screened using a null-biosensor. Only five compounds displayed Δτ > 50 ps, indicating that our method for eliminating fluorescent compounds removes nearly all false positives. These five compounds were also excluded from further consideration ( Supplementary Fig. S1A). (C) and (D) Plots of ΔFLT vs. Δ(G/R) show excellent, reproducible correlation between the two measurements for the 2CS biosensor (C), but distinct from those observed for the null-biosensor (D), indicating that the hit compounds induce a structural change in the 2CS biosensor.  Fig. 4 and 7C, Fig. 5 and 6B, and Table 1).
Classification of compounds by physicochemical characteristics. The 18 hit compounds were subjected to cheminformatic analysis, to determine whether any shared common chemical scaffolds. Compounds with a Tanimoto coefficient and maximum common substructure (MCS) 36 scores above 0.4 were binned as clusters, while those with scores below 0.4 were classified as singletons. The analysis yielded diverse scaffolds 36,37 of hit compounds (Supplementary Fig. S2 and Supplementary Table S1).

Discussion
We have identified new compounds based on an increase in ΔFLT within a human cardiac 2CS biosensor expressed in live mammalian cells at low [Ca 2+ ] (the normal condition in the cytoplasm of HEK cells). This decrease in FRET implies that the actuator (A) and nucleotide-binding domains (N) of SERCA2a moved apart, supporting an open configuration at low [Ca 2+ ] in HEK cells, possibly priming SERCA in a more open state to bind Ca 2+ . Previous studies with a different SERCA2a biosensor indicated that the addition of Ca 2+ induces a decrease in FRET 21 . Our functional assays at high and mid [Ca 2+ ] show activating, uncoupling, and inhibiting effects that may correlate with structural changes. Further future elucidation of the compounds' effects on SER-CA2a conformational states will require detailed analysis of FLT-detected FRET and transient kinetics data. We identified three categories of activators that (1) increase both Ca 2+ -ATPase activity and Ca 2+ -transport to increase CR (Compound 7, Fig. 4) (2) increase Ca 2+ -ATPase activity and Ca 2+ -transport to decrease CR (Compounds 2, 4,  8, and 9, Fig. 5), and (3) increase Ca 2+ -ATPase activity but inhibits Ca 2+ -transport to decrease CR (Compounds 1,  Table 1 for panels C, D, and E. Data are presented as mean ± SEM, n = 3, *p < 0.05. www.nature.com/scientificreports/ 3, 5, and 6) (Fig. 6). We identified four subcategories of inhibitors based on the extent of decrease in Ca 2+ ATPase activity and Ca 2+ -transport for SERCA2a (Table 1, Fig. 7A). Most FRET-EC 50 values were smaller (higher potency) for activators (Compounds 1-9; 0.3-7 μM) than for inhibitors (Compounds 10-18; (3-32 μM) (Table 1). However, the functional C 10 and EC 50 values were smaller, indicating greater potency, for inhibitors than for activators. Potencies observed by FRET and function are not precisely correlated, probably because the assays were performed on different types of samples (live cells vs. purified proteins), low nM [Ca 2+ ] in live HEK cells 43 vs. μM [Ca 2+ ] in the pCSR in vitro assays), which measure different properties (structure vs function). Functional CRC assays showed that inhibitors tend to induce larger changes (indicating higher efficacy) than activators, in both Ca 2+ -ATPase activity and Ca 2+ -uptake. Most inhibitors induced a larger change in Ca 2+ -uptake than in Ca 2+ -ATPase activity, decreasing CR.

Scientific Reports
Most of the activators reduced CR, inducing larger changes in Ca 2+ -ATPase activity than in Ca 2+ -uptake. A notable exception is Compound 7, which increases Ca 2+ -transport even more than it increases Ca 2+ -ATPase activity, increasing CR. This compound will be a high priority as a lead compound for future efforts in medicinal chemistry and assays of physiological function. Compounds 2, 4, 8, and 9 will have only slightly lower priority.
Ten compounds were binned into four clusters (A-D); eight were singletons (E-L) ( Table 1). Many compounds showed similar functional traits, suggesting that ligand-sensing sites in SERCA2a are recognized by a range of scaffolds, or that these sites are close to each other, providing potentially powerful tools in the design of future compounds [44][45][46] . Only Compound 7 (singleton F) induced higher activation in Ca 2+ transport than in the Ca 2+ -ATPase activity. Compounds 2 (cluster A), 4 (cluster B), 8 (singleton G), and 9 (cluster C) induced similar effects of moderate activation of Ca 2+ -ATPase activity, with smaller activation of Ca 2+ -transport. Compounds in activator clusters A (Compounds 1 and 3) and B (Compound 5) along with Compound 6 from singleton E, showed similar functional effects: moderate ATPase activation with mild inhibition of Ca 2+ -transport at [Ca 2+ ] MAX . Inhibitor compounds in clusters D (11, 12, and 13), C (10), and H-L (14-18) induced a range of effects at both [Ca 2+ ], which will be useful in designing derivatives for structure activity relationship (SAR) analysis.
There was negligible overlap in hit compounds identified in our previous FRET-HTS of the DIVERSet-CL targeting tumor necrosis factor receptor 1 27 . There was 81% overlap in the fluorescent compounds detected (and thus rejected) in these two HTS studies, indicating that our FRET-HTS methodologies are effective and versatile 26,29 . In our previous study of the DIVERSet-CL, using the SERCA2a Ca 2+ -ATPase activity as the primary HTS assay (ATPase-HTS), we discovered 19 activators 30 . While no identical activators were found in that ATPase-HTS study 30 and in the current FRET-HTS study, there were several compounds with similar scaffolds that showed similar functional results. These compounds share the oxadiazol scaffold and activated the Ca 2+ -ATPase activity but inhibited Ca 2+ -uptake. Another common scaffold is the amide group; six compounds 30 identified with an amide induced a smaller increase in the Ca 2+ -transport than in the Ca 2+ -ATPase activity, reducing CR similar to Compound 9 in this study. It is not surprising that the two studies did not identify the same compounds, because (a) the FRET-HTS assay was performed with human cardiac 2CS in live HEK cells in low [Ca 2+ ], while the ATPase-HTS assay was done in purified SR from rabbit skeletal muscle (SERCA1a) under high [Ca 2+ ] 30 , (b) the FRET assay is much more precise than the functional assay, (c) the relationship between SERCA structure and function is complex, and (d) the binding sites on SERCA2a for these compounds are unknown. As discussed above, a ligand-binding site may recognize several different scaffolds [44][45][46] . It is also possible that a compound binds to PLB or competes with PLB for binding to SERCA, thus increasing SERCA2a activity, as was shown for the activator, istaroxime 47,48 . These observations highlight the value of complementary HTS assays for the same target.
Activation of Ca 2+ -transport by SERCA2a is needed when cardiac relaxation is impaired, as in diastolic dysfunction 1 or diabetic cardiomyopathy 49 . SERCA2a activation is a promising strategy, in combination with current drugs such as β-blockers and ACE inhibitors 50 . Activation of SERCA also has therapeutic potential for Alzheimer's disease 51 or Duchenne muscular dystrophy (DMD) 52 . Until recently, very few compounds were known to stimulate SERCA2a: CDN1163 (stimulates Ca 2+ transport) 24,53 , CP-154526 (increases the apparent Ca 2+ affinity of SERC2a) 54 , Ro 41-0960 (increases SERCA2a maximal activity in high Ca 2+ ) 54 , and istaroxime (stimulates SERCA2a activity) 55 . However, our recent ATPase-HTS assay identified ~ 19 new activators of SERCA 30 , and we identified nine in the present study. A SERCA activator from our previous work (CDN1163) shows promise as a therapeutic agent for Alzheimer's disease 51 and for DMD 52 . Of all these SERCA2a activators, only istaroxime has been in phase IIb clinical trials for treatment of heart failure 55,56 . However, because of its unsuitability for human usage 56 , istaroxime must be modified 47,48 .
Compounds 1, 3, 5, and 6 induced small effects on the Ca 2+ -ATPase activity (~ 10-25% increase) and induced a negative effect on the Ca 2+ -transport ( Fig. 6C and D), thus decreasing the CR, which is likely to increase heat output 15,16,57 . These effects are similar to that of SLN on SERCA1a (skeletal muscle), where SLN reduces Ca 2+ -transport without affecting the Ca 2+ -ATPase activity (SERCA1a uncoupling), thus reducing CR 15 . Uncoupling of SERCA1a leads to higher usage of ATP, which enhances non-shivering thermogenesis (NST) 15 . Another contributor to NST is Ca 2+ leak from SR through resting RyR channels, stimulating SERCA to re-sequester Ca 2+ into SR, thus using more ATP and generating heat 58 , which has been suggested as a potential therapeutic strategy for reducing obesity 15,57 .
Six decades of research for SERCA inhibitors as oncology therapeutics have yielded hundreds of SERCA inhibitors with varying potencies and efficacies 17 . Similarly, our discovery of new SERCA inhibitors with a range of potencies and efficacies is likely to be advantageous for non-cardiac applications 17,18 .
Here we successfully used the 2CS biosensor to identify novel small-molecule effectors of SERCA with diverse chemical scaffolds, resulting in an array of activator and inhibitor hit compounds. Most importantly, based on the amplitude of the functional effects on SERCA2a, we discovered a potential lead compound (Compound 7) that activates Ca 2+ -uptake more than the Ca 2+ -ATPase activity, increasing the CR, so this will be a high priority for future efforts in medicinal chemistry and assays of physiological function, along with four other promising www.nature.com/scientificreports/ SERCA2a activators. The innovative technology included two novel plate-readers -the FLT instrument used in the primary screen, and a spectral instrument -that were used to remove compounds with interfering fluorescence signals, allowing us to focus on valid SERCA activators and inhibitors. It is possible that some of the eliminated fluorescent compounds have potential as SERCA2a effectors, which could be evaluated in future work using our ATPase activity assay 30 . In future studies, we will evaluate these hit compounds in more functional detail, including the full range of [Ca 2+ ], SERCA isoforms, Na + /Ca 2+ exchanger, RyR, and L-type Ca 2+ -channels. Medicinal chemistry will be done to elucidate SAR and to design analogs with greater potency and specificity 27,59 , justifying studies in intact muscles and animals. Finally, we have shown that our primary screening technology can perform precise HTS on several thousand compounds per hour, making this approach capable of application on an industrial scale, screening millions of compounds.

Molecular biology.
A two-color intramolecular human SERCA2a (2CS) biosensor, based on human cardiac SERCA2a fused to green fluorescent protein (eGFP) and red fluorescent protein (tagRFP) was developed to detect structural changes that are related to the functional changes of SERCA 20,22,25 . Briefly, tagRFP was genetically fused to the N-terminus of SERCA (A-domain) and eGFP was inserted as an intrasequence tag before residue 509 in the nucleotide-binding domain (N-domain) 60,61 . Donor-only and acceptor-only (1CS) biosensors were created in a similar manner as the 2CS biosensor but with the construct containing either only eGFP or only tag-RFP, respectively. The fluorescent proteins fused to SERCA in 2CS and 1CS do not significantly affect SERCA activity, in membranes purified from HEK cells 23,25 . A null-biosensor construct consisting of eGFP and tagRFP connected by a 32-residue unstructured flexible linker peptide (G32R) was created as described previously 23,25 . All constructs were cloned into expression vectors containing the genes for antibiotic resistance to G418, puromycin, or blasticidin. Compound handling. A 50,000 DIVERSet-CL was purchased from ChemBridge Corporation (San Diego, CA) at a 10 mM stock concentration for each compound. All compounds met the high quality standard of 100% identification by NMR and/or LC-MS and have a minimum purity of 85% and their identity verified using LC-MS/ELSD as confirmed by the ChemBridge Corporation. For the FLT HTS initial screens, the compound library was reformatted into 384 well Echo compatible plates using the Biomek FX (Beckman Coulter, Miami, FL) and 5 nL of either compound (columns 3-22 and 27-46) or DMSO (columns 1-2, 23-26, and 47-48) was dispensed into forty 1536 well black polystyrene assay plates (Greiner, Kremsmünste, Austria) using an Echo 550 liquid dispenser (Beckman Coulter) to yield a final assay screening concentration of 10 μM. The low autofluorescence and low interwell cross-talk of these plates made them advantageous for FLT measurements. Plates were heat sealed with a PlateLoc Thermal Microplate Sealer (Agilent, Santa Clara, CA) and stored at − 20 °C prior to use. The same methods were applied for subsequent FLT retesting of the hit compounds identified in the FLT screen (Fig. 1B, steps 1-3), except that the [compound] was tested at 10 μM and 30 μM in triplicate, where the latter gave more reproducible results. FRET CRC assay plates (0.78-100 μM compound range) with at least ten different compound concentrations were made by adding the appropriate volume of compound or DMSO into black 384 well plates (Greiner Bio-One) using a Mosquito HV (SPTLabTech, United Kingdom). Subsequent Ca 2+ -ATPase activity and Ca 2+ -transport CRC assay plates (0-50 μM compound range) with repurchased compounds were made in a similar manner using with the Echo 550 (Beckman Coulter) using either 384 well transparent plates (Greiner Bio-One) or black-walled plates with transparent bottoms (Greiner Bio-One), respectively.

HTS sample preparation and FRET measurements.
On each day of screening, cells were harvested, washed three times with PBS, and centrifuged at 300 g for 5 min. Cells were filtered using a 70 µm cell strainer and diluted to 1-2 × 10 6 cells/mL. Cell concentration and viability were assessed using the Cell countess (Invitrogen) and trypan blue assay. During assays, cells were constantly and gently stirred using a magnetic stir bar at room temperature, keeping the cells in suspension and evenly distributed to avoid clumping. HEK 293cells expressing 2CS were dispensed at 5 μL or 50 μL per well into assay plates (dispensed into 40 assay plates, each containing 1536 wells) pre-plated with compounds (from a DIVERset 50,000 compound library) using a Multdrop Combi liquid dispenser (Thermo Fisher Scientific, Pittsburg, PA) and sealed until needed. Because the kinetics of membrane permeability, diffusion, and/or binding of the compound to live cells may be compound-dependent, we tested two incubation times, 20 min and 120 min, for the FRET CRC. FRET EC 50 values determined from both incubations were similar, but the 120 min incubation yielded a more reproducible and sigmoidal curve. Plates containing eight-point concentration curves of three tool compounds (known SERCA www.nature.com/scientificreports/ effectors thapsigargin, BHQ, and CPA) 22 were used as positive controls for biosensor function and performance prior to running the full-scale FRET-HTS assay. The primary FLT-FRET-HTS assay was performed over two days with a custom HTS fluorescence lifetime plate reader (FLT-PR) with donor emission detected at 517 nm (Fig. 1B, step 1). Initial hits from this FLT screen were selected (using rZ-score, discussed below), then fluorescent compounds were removed using the Similarity Index (SI) calculated from the spectral measurement acquired with the SUPR (SI, discussed below) (Fig. 1B, step  2). Both instruments were provided by Photonic Pharma LLC (Minneapolis, MN) 23 .
The same methods were applied for subsequent FRET retesting (Fig. 1B, step 4) of the reproducible hit compounds identified in Fig. 1B, steps 1-3, except that the compounds were tested at 10 μM and 30 μM [compound]. 160 hit compounds were selected from the library master plates and reloaded onto new assay plates for retesting with 2CS and a null-biosensor, using FLT-PR (ΔFLT) and SUPR (Δ(G/R)). This step was designed to remove compounds that bind directly to the fluorescent protein or produce other artifacts in the FLT reading that do not involve FRET. Then 18 hit compounds, representing a range of ΔFLT, were selected and purchased from ChemBridge to determine CRC from FRET, Ca 2+ -ATPase activity, and Ca 2+ -transport assays using at least ten different concentrations by repeatedly scanning the 1536-well plates.

FRET-HTS instrumentation and data analysis.
A detailed description of the high-throughput fluorescence lifetime plate reader (FLT-PR) and spectral unmixing plate reader (SUPR), manufactured by Fluorescence Innovations Inc and provided by Photonic Pharma, LLC was described previously 23,26 . Briefly, for lifetime measurement with the FLT-PR, the observed donor-fluorescence waveform, I(t) was fit by a convolution of the measured instrument response function (IRF) (Eq. 1a) and a single-exponential decay F(t) to obtain the lifetime (τ) of the donor fluorophore 22,26,62 in the absence (τ D ) and presence (τ DA ) of the acceptor as described in Eq. (1b): In experiments with a donor-only control, FRET efficiency (E) was determined as the fractional decrease of donor FLT in the absence and in the presence of acceptor as in Eq. (2): E was determined in the presence and absence of compound and normalized relative to E of the DMSO control. For spectral detection of FRET with SUPR, the observed fluorescence emission spectrum F(λ) was fit by least-squares minimization of a linear combination of component spectra for donor (G for green), acceptor (R for red), cellular autofluorescence (C) and water Raman (W), as described previously 22 . The change in ratio of the mole fractions of the G and R component spectra between compound and DMSO control (Δ(G/R)) provides a direct indication of a change in FRET due to biosensor structural changes, independent of the lifetime measurements. Together, these complementary metrics provide an effective method for eliminating false positives arising in either method.
HTS data analysis. FLT-PR data was used as the primary metric for flagging potential hit compounds.
After fitting waveforms with a single exponential decay to quantify donor lifetime, the change in fluorescence lifetime due to compound (Δτ) was computed by performing a moving median subtraction in the order the plate was scanned, with a window size of 24 wells, rather than subtracting DMSO controls. The reasons for this are twofold: 1) plate gradients are often observed due to heating of the digitizer during acquisition and 2) performing Δτ computations with DMSO controls can sometimes result in artifacts as a half of the DMSO wells are on the edge of plates, which occasionally exhibit artifacts due to processes needed for the preparation of the compound library being tested. As most compounds are likely to be non-hits, and therefore DMSO like, computation of a moving median is an effective alternative to solving both gradient issues and edge-effect distortion of the primary metric for hit selection, Δτ.
Previous validation in 1536-well plates indicated that the Z' parameter -a measure of HTS assay quality that factors in the signal window and the variance of positive and negative controls (i.e., thapsigargin and DMSO vehicle) 63 -yielded a value of 0.62 25 . A value of 0.5 ≤ Z' < 1 indicates excellent assay quality, ready for large-scale HTS 63 as we recently showed for an ATP-based HTS assay 30 . Data from the control (tool) compounds was not needed for assessment of assay quality in the full-scale HTS in this study, instead we used the coefficient of variance (CV), computed using wells containing only DMSO, to assess the quality of the plates, in order to separate within-plate and between-day variability, as in our previous ATP-based HTS study 30 .
In our previous ATPase-based HTS study, we defined a hit compound as one that changes the ATPase signal by 4SD relative to the DMSO controls 30 . Given the increased precision (< 1% CV, Fig. 2A) afforded by the FLT measurement and the availability of complementary HTS measurements using the spectral plate reader on the same plate for further triage, we set our hit threshold to an rZ-score of ± 3, on a plate-by-plate basis, in order to include a broader range of initial hit compounds. The rZ-score was used (instead of the standard Z-score), where the median (M) and median absolute deviation (MAD) are used in place of the mean and standard deviation (Eq. 3), to best capture the most hits, as the standard Z-score is more susceptible to strong outliers 23 .
(1a)  22 was computed by comparing a region (500-540 nm) of the donor-only spectrum (I (a) ) for each well to that of the plate-wide average DMSO spectrum (I (b) ) in the same wavelength band as described in Eq. 4 26 . Compounds that reproducibly exceeded an SI rZ-score of 5 (corresponding to an SI of 2 × 10 -4 ) were deemed likely fluorescent compounds and were removed from consideration. Spectral (SUPR) data was processed similarly to FLT-PR data, with the Δ(G/R) metric being computed by applying the same moving median filter on the initial measurement of the ratio of donor (G) to acceptor (R) mole fractions (G/R) 21,25 . The hit threshold was also set using an rZ-score of ± 3. While the FLT-PR data and SUPR data showed strong correlation, the FLT-PR data exhibited some clear outliers, presumably due to compounds directly modifying the donor lifetime. To eliminate these likely interfering compounds, correlation was enforced by eliminating compounds that exceed an rZ-score of ± 3 from the median value of the ratio of Δτ over the Δ(G/R) metric.
Cardiac SR preparation. Cardiac SR vesicles were isolated from fresh porcine left ventricular tissue using differential centrifugation of the homogenized tissue as previously described 20 . The SR vesicles were flash-frozen and stored at − 80 °C until needed. The SERCA concentration in the ER preparations purified from HEK cell homogenate is at least 10 times less than in purified porcine cardiac SR 20 , but there was sufficient expression of the fluorescent SERCA2a biosensor in HEK cells to detect FRET with high precision by FLT 22,23,26 . Effects of hit compounds on the Ca 2+ -ATPase activity of SERCA . Functional assays were performed using porcine cardiac SR (pCSR) vesicles 20 . An enzyme-coupled, NADH-linked ATPase assay was used to measure SERCA ATPase activity in 384-well microplates. Each well contained 50 mM MOPS (pH 7.0), 100 mM KCl, 1 mM EGTA, 0.2 mM NADH, 1 mM phosphoenol pyruvate, 10 IU/mL of pyruvate kinase, 10 IU/mL of lactate dehydrogenase, 7 µM of the calcium ionophore A23187 (Sigma), and CaCl 2 was added to set free [Ca 2+ ] to three different concentrations 23 . Ca 2+ -ATPase activity was measured at three [Ca 2+ ]: [Ca 2+ ] MAX (saturating, pCa 5.4), [Ca 2+ ] MID (subsaturating, midpoint, pCa 6.2), and [Ca 2+ ] BAS (basal non-activating, pCa 8.0). 10 μg/mL of SR vesicle, calcium, compound (0.048 to 50 μM), and assay mix were incubated for 20 min at room temperature before measurement of functional assays with each of the 18 hit compounds, because a shorter incubation time than the FRET live-cell assays achieved optimal responses. The assay was started upon the addition of MgATP, at a final concentration of 5 mM (total volume to 80 μL), and absorbance was read at 340 nm in a SpectraMax Plus 384 microplate spectrophotometer (Molecular Devices, Sunnyvale, CA).
Effects of hit compounds on the Ca 2+ -transport activity of SERCA . Ca 2+ -transport assays were performed with similar porcine SR samples as in the Ca 2+ -ATPase assays described above. The compound effect on the Ca 2+ -transport activity of SERCA2a was determined using an oxalate-supported assay in which the change in fluorescence in a Ca 2+ -sensitive dye, Fluor-4, was determined as previously described 23 . A buffered solution containing 50 mM MOPS (pH 7.0), 100 mM KCl, 30 mg/mL sucrose, 1 mM EGTA, 10 mM potassium oxalate, 2 M Fluo-4, 30 µg/mL porcine cardiac SR vesicles, CaCl 2 calculated to reach the free [Ca 2+ ] (pCa 8.0, 6.2, and 5.4), and compound (0.048 to 50 μM) was dispensed into 384-well black walled, transparent bottomed plates (Greiner Bio-One) containing the tested small molecule and incubated at 22℃ for 20 min while covered and protected from light. To start the reaction, MgATP was added to a final concentration of 5 mM, and the decrease in 485-nm excited fluorescence of Fluo-4 was monitored at 520 nm for 15 min using a FLIPR Tetra (Molecular Devices, San Jose, CA). Data analysis of FRET CRC assays of hit compounds. FRET efficiency (E) (Eq. 2) was determined as the fractional decrease of donor lifetime (τ D = 2.5 ± 0.01 ns for 1CS, donor only) in the presence of acceptor (2CS, τ DA = 2.33 ± 0.001 ns) due to FRET. E was plotted as "FRET Effect (E/E DMSO )" vs [compound], fitted to the Hill's function for determination of FRET-EC 50 27,31 . This normalization of E corrects for variation of controls done on different days. Data analysis of Ca 2+ -ATPase and Ca 2+ -transport activities from CRC assays. SERCA2a activity, F (rate of ATP hydrolysis or Ca 2+ uptake), was measured at varying pCa and varying compound concentration. F measured at [Ca 2+ ] MAX (saturating, pCa 5.4) or [Ca 2+ ] MID (subsaturating, midpoint, pCa 6.2) was corrected by subtracting the basal rate at pCa 8.0) and the % effect due to compound was reported. Concentration response curves (CRC) were fitted using the Hill function to determine V MAX (the activity at saturating [compound]), and EC 50 , the compound concentration at 50% effect 54 . When the CRC did not achieve saturation, the maximal change (Δ) in activity was determined, to yield ΔF MAX and ΔF MID at the [Ca 2+ ] MAX and [Ca 2+ ] MID conditions, respectively (Fig. 5C, Table 1). ΔF MAX, ΔF MID , C 10 (compound concentration inducing 10% effect), and EC 50 are reported in Table 1.